ChIC/CUT&RUN-seq

Last edited Tue 25 Apr 2023

Chromatin Immuno-Cleavage/Cleavage Under Targets and Release Using Nuclease (ChIC/CUT&RUN) is an emerging chromatin profiling technique used to analyze DNA-protein interactions^1,2. While ChIP-seq uses sonication of fixed cells to fragment chromatin, ChIC/CUT&RUN-seq uses an enzyme (pAG-MNase).

The pAG-MNase enzyme is conjugated to an antibody for proteins of interest. Chromatin fragments containing proteins of interest can then be purified using immunoprecipitation techniques. After this, the DNA fragments are purified and sequenced. The sequencing results can be used to determine the regions of DNA your protein of interest interacts with.

As outlined in the protocol below, Abcam provides the pAG-MNase enzyme.

Stage 1 - Cell harvesting and bead preparation

Materials required

• ConA magnetic beads (10 µL per reaction)
• PBS
• Cell sample (2.5 x 10⁵ per reaction)
• Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail)
• Binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂ , 1 mM MnCl₂)
• Centrifuge
• Magnetic rack

Steps

Harvest cells and suspend in 3–4 mL ice-cold PBS.

Count cells and take the desired number of cells per reaction.

• We use 2.5x10⁵ HeLa cells per reaction. However, this may need optimizing for each cell line.
• See our counting cells using a hemocytometer protocol.

Prepare wash buffer.

Add protease inhibitor cocktail (eg ab65621) before use.

Wash cells three times in wash buffer.

• Add 1 mL of wash buffer to cells.
• Spin down cells at 600 x g for 3 min.
• Remove supernatant and repeat for a total of three washes.

Prepare a slurry of conA magnetic beads in binding buffer.

• Take 10 µL of beads per reaction.
• Wash beads in 1 mL of binding buffer on the magnetic rack.
• Resuspend the beads in 10 µL per reaction of binding buffer.

Bind cells to activated beads.

• Mix cells and beads together for 20 min at room temperature, mixing gently every 4 min.

Separate the cell-bound beads from solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Stage 2 - Permeabilization and binding of antibodies

Materials required

• Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail)
• 0.5 M EDTA stock solution
• 5% Digitonin stock solution
• Antibody buffer (see Step 1)
• Antibodies validated for ChIC/CUT&RUN-seq
• Horizontal gyratory shaker
• Magnetic rack

Steps

Prepare the antibody buffer.

• Dilute EDTA and Digitonin stock solutions in the Wash Buffer to include final concentrations of 0.2 mM EDTA and 0.05 % Digitonin.
• Before use, add protease inhibitor cocktail (ab65621) and Digitonin (ab141501).

Resuspend beads in antibody buffer.

• Resuspend in around 100 µL per reaction and aliquot out into separate tubes if prepping serval reaction in same tube.

Add antibody to bead mix.

• Recommended dilutions will always be suggested on the antibody datasheet
• Incubate overnight at 4 °C in a horizontal shaker.

Stage 3 - Binding of pAG-MNase

Materials required

• pAG-MNase (ab285373)
• Wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.05 % Digitonin)
• 5 % Digitonin stock solution
• Digitonin wash buffer (see step 2)
• Horizontal gyratory shaker
• Magnetic rack

Steps

Separate the antibody-bound beads from the solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Prepare Digitonin wash buffer.

• Dilute Digitonin stock solution in the wash buffer to give a final concentration of 0.05 % Digitonin.
• Before use, add a protease inhibitor cocktail (ab65621) and Digitonin (ab141501).

Wash beads twice with Digitonin wash buffer.

• Add 200 µL of Digitonin wash buffer to the beads per reaction
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.
• Repeat for a total of two washes.

Incubate the beads with pAG-MNase solution.

• Resuspend beads in 50 µL of digitonin wash buffer.
• Add pAG-MNase to a final concentration of 700 ng/µL.
• Incubate for 1 – 2 h at room temperature with horizontal shaking at 130 rpm.

Stage 4 - Chromatin-targeted digestion

Materials required

• Low salt buffer (20 mM HEPES pH 7.5, 0.05 % Digitonin, 0,5 mM Spermidine)
• Incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl₂, 0.05 % Digitonin)
• Magnetic rack

Steps

Separate the antibody-bound beads from the solution.

• Place the tube on a magnetic rack for 1 – 2 min.
• Discard supernatant and keep beads.

Prepare low salt, incubation and wash buffers.

• Add Digitonin (ab141501) before use.
• Place buffers on ice.

Wash beads twice with Digitonin wash buffer.

• Add 200 µL of Digitonin wash buffer to the beads per reaction and incubate for 5 min.
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.
• Repeat for a total of two washes.

Wash beads with low salt buffer.

• Add 200 µL of low salt buffer to the beads per reaction and incubate for 5 min.
• Place the tube on the magnetic rack for 1 min.
• Discard supernatant and keep beads.

Digest chromatin by incubating beads with ice-cold incubation buffer.

• Add 100 µL of incubation buffer and incubate for 15 min on ice, mixing halfway through the incubation.
• Place the tube on the magnetic rack for 1 min.
• Discard the supernatant and keep beads.
• The incubation buffer contains Ca²⁺ ions, which activate the pAG-MNase enzyme to cleave DNA. You may need to optimize the incubation time.

Stage 5 - Elution of DNA and sequencing

Materials required

• Stop buffer (20 mM EGTA, 170 mM NaCl, 0.05 % Digitonin, 50 µg/mL RNAse A, 25 µg/mL Glycogen)
• Thermo-mixer
• Magnetic rack
• Commercial extraction, quality control, and library preparation kits

Steps

Incubate beads with stop buffer.

• Add 100 µL of ice-cold stop buffer.
• Incubate beads for 30 min at 37 °C in a thermomixer, shaking at 700 rpm.

Collect fragments.

• Place the tube on the magnetic rack for 1 min.
• Aliquot the supernatant into new tubes.

Extract DNA and prepare for sequencing.

• Use commercial extraction, quality control, and library preparation kits according to the manufacturer’s instructions.

Send DNA fragments for sequencing.